DNA polymerases are enzymes which are useful in many recombinant DNA techniques, such as nucleic acid amplification by the polymerase chain reaction (“PCR”), self-sustained sequence replication (“3 SR”), and DNA sequencing. Thermostable DNA polymerases are particularly useful. Because heat does not destroy the polymerase activity, there is no need to add additional polymerase after every denaturation step.
In its catalytic cycle, the DNA polymerase-DNA complexes formed are known to undergo a rate-limiting, conformational transition from an ‘open’ to ‘closed’ state, upon binding of the ‘correct’ dNTP or ddNTP at the active site. In the ‘closed’ state, Mg2+ (or other metal ion) mediates a rapid chemical step involving nucleophilic displacement of pyrophosphate by the 3′ hydroxyl of the primer terminus. The enzyme returns to the ‘open’ state upon the release of pyrophosphate (PPi) and translocation initiates the next round of reaction. While the ternary complex (Enzyme-DNA-dNTP (or ddNTP) can form in the absence of Mg2+ (or other metal ions), it is proficient in chemical addition of nucleotide only in the presence of Mg2+ (or other metal ions). Mg2+ (or other metal ion)-deficient conditions tend to lead to non-covalent (physical) sequestration of first ‘correct’ dNTP in a tight ternary complex (Doublie et al. (15 Feb. 1999) Structure Fold. Des., 7(2):R31-5).